The present invention relates to a method of breeding tomatoes with increased starch content in the young fruit and subsequently increased soluble solids content in the mature fruit. In addition, it relates to the use of genes that increase starch in the tomato.
The solids content of ripe tomato fruit is a major determinant of its quality. Increasing the soluble solids (largely sugars and organic acids) content and thereby improving the value of industry tomatoes and the taste of fresh market tomatoes have been the goal of research projects for many years. Several approaches to improving solids levels have been taken, encompassing both agrotechnical and genetic manipulations.
Soluble solids content of tomato fruit are primarily comprised of sugars, organic acids and salts. Collectively the soluble solids content is a major determinant of fruit quality, both for industry use and for fresh market consumption. Approximately half of the soluble solids content is contributed by the sugar fraction which, in all standard cultivars of Lycopersicon esculentum, consists of the monosaccharide reducing sugars glucose and fructose in approximately equimolar concentrations.
Several strategies to increase sugar concentration in ripe tomato fruit have been explored. Genetic manipulations include the transfer of undefined traits of high soluble solids from wild species of Lycopersicon (Rick C. M. 1974. Hilgardia 42:493-510; and Hewitt J. D., Dinar M. and Stevens M. A. 1982. J. Am. Soc. Hort Sci. 107:896-900) and more recently the transfer of the genetic trait of sucrose accumulation from the wild Lycopersicon chmielewskii (Yelle S., Hewitt J. D., Robinson N. L., Damon N. S. and Bennett A. B. 1988. Pl. Physiol. 87:737-740; and Yelle S., Chetelat R. T., Dorais M., Deverna J. W. and Bennett A. B. 1991. Pl. Physiol. 95:1026-1035.) and L. hirsutum (Miron D. and Schaffer A. A. 1991. Pl. Physiol. 95:623-627), as well as the transfer of the genetic trait of high fructose to glucose ratio in the mature fruit, from L hirsutum (U.S. patent application Ser. No. 08/530,216, the disclosure of which is incorporated herein by reference). The latter approach was made possible by the study of the components of carbohydrate metabolism in developing tomato fruit tissue with the purpose of identifying biochemical steps whose modification may lead to increased soluble carbohydrate content in the fruit (Yelle et al., 1988, 1991; Miron and Schaffer, 1991). Once identified, these biochemical processes could then be targeted for modification by classical genetic means, assisted by selection for the genotypic biochemical trait, or by molecular genetic strategies.
The young developing tomato fruit is characterized by a transient starch accumulation which can contribute over 25% of the dry weight of the fruit tissue. Starch concentration begins to increase within days after anthesis and reaches peak concentrations before the mature green stage (Schaffer, A. A. and Petteikov, M. 1997a. Plant Physiology 113:739-746). At the mature stage this starch is practically absent in the tomato fruit tissue. It has been hypothesized that the transiendy accumulated starch serves as a reservoir of carbohydrate for the later accumulation of soluble sugars in the mature fruit (Dinar M. and Stevens M. A. 1981. J. Am. Soc. Hort. Sci. 106:415418). Dinar and Stevens laid the groundwork for this hypothesis in their study comparing seven genotypes of tomato whose total soluble solids (TSS) values in the ripe fruit spanned the spectrum from 4.6 to 6.3 Brix. They found that TSS values in ripe fruit were positively correlated with starch content in young, immature fruit and proposed that the products of starch hydrolysis contribute to the accumulation of soluble sugars.
The tomato plant translocates photosynthate to the fruit in the form of sucrose (Walker L. J. and Ho L. C. 1977. Ann. Bot. 41:813-823) and therefore, the temporal accumulation of starch will presumably be determined by temporal changes in the activities of key enzymes involved in sucrose to starch metabolism. The enzymatic pathway of starch synthesis in young tomato fruit has been studied and described (Schaffer, A. A. and Petreikov, M. 1997a. Plant Physiology 113:739-746; Schaffer, A. A. and Petreikov, M. 1997b. Physiologia Plantarum 101:800-806). Four enzymes were identified Mat potentially limit starch accumulation in these fruit, based on their absolute activities, as well as on the developmental changes in their activities which correlate temporally with the developmental changes in starch levels. These enzymes include those that catalyze the initial steps of sucrose metabolism in the young fruit (sucrose synthase, E.C. 2.4.1.13, and fructokinase, E.C. 2.7.1.4) as well as the latter steps of starch synthesis (ADP-glucose pyrophosphorylase, E.C. 2.7.727, and starch synthase, E.C., 2.4.1.21). In addition, Schaffer and Petreikov have shown that starch accumulation is tissue specific, localized primarily in the columella and inner pericarp tissues, and suggested that relative contributions of these tissues to fruit bulk could impact on Suit starch content.
Research has clearly shown that one of the above mentioned enzymes, ADP-glucose pyrophosphorylase (ADPGPPase), may be limiting to starch synthesis in tomato fruit, as well as in other starch accumulating tissues, such as potato tubers. In Stark D. M., Barry G. F., and Kishore G. M. 1996. Ann. NY Cad Sci 792:26-36, transgenic tomato plants and potato plants were developed with a bacterial mutant form of ADPGPPase (E. coli, GlgC16, a glycogen overproducer). Transgenic tomatoes showed a higher starch content in the immature fruit and an increased sugar content in the mature fruit Transgenic potato tubers with the same bacterial gene construct also showed an increase in starch content. Reciprocally, inhibition of ADPGPPase activity decreased the starch content of transgenic potato tubers, further indicating the importance of ADPGPPase in controlling starch accumulation.
The use of a gene for ADPGPPase of bacterial origin requires molecular genetic manipulations in order for the gene to function in eucaryotic plant tissue. For example, it requires that an artificial gene construct be developed that will encode a fusion polypeptide containing a specific amino terminal Lot peptide, not present in the procaryotic gene, as well as other DNA sequence additions that will cause in plant cells transcriptional termination, and the addition of polyadenylated nucleotides to the 3xe2x80x2 end of the RNA sequence. In comparison, the use of a plant gene for similar transformations does not require these manipulations. In addition, the development of plants with increased or modified activity of these enzymes, based on the natural transfer through classical breeding techniques of naturally occurring alleles of these genes, can benefit from a number of advantages. For example, classical breeding techniques lead to the positioning of the desired allele in the natural position of the gene of interest, leading to genetic stability and obviating the unpredictable xe2x80x9cpositionxe2x80x9d effects characteristic of the development of transgenic organisms. In addition, with respect to consumer preferences, there are obvious advantages of a naturally derived commercial product such as a tomato fruit, compared to a transgenically derived tomato fruit.
With resect to fructokinase, two genes from tomato fruit have been identified, cloned and sequenced (Kanayama, Y. et al. 1997. Plant Physiology 113:1379-13S4). One of these genes, FK2, is particularly involved in the metabolic pathway associated with starch synthesis (Kanayama et al. 1998. Plant Physiology 117:85-90). Similarly, the gene for sucrose synthase from tomato fruit has been cloned and sequenced (Wang, F., et at. Plant Physiology 103:1463-1464; ) and has been shown to be the gene for sucrose synthase of sink tissue (Fu, H. and Park, W. D. Plant Cell 7:1369-1385).
With respect to ADPGPPase, the enzyme functions in higher plants as a heterotetramer, comprised of two large and two small subunits (Preiss, J. and Sivak, M. In: Photoassimilate Distribution in Plants and Crops, Zamski, E. and Schaffer, A. A., eds., Marcel Dekker Publ, NYC, pp.63-96, 1996) which are under independent genetic control. Three separate L. esculentum genes coding for the large subunits and one gene for the small subunit have recently been cloned and sequenced (Chen, B. Y. and Janes, H., 1995, Plant Physiology 109.1498; Park, S. W. and Chung, W. I. 1998. Gene 206.215-221). Much effort has been made in order to identify sources of ADPGPPase genes in plants that may contribute to improving starch content, as for example in corn (Giroux, M. J. et al., Proc. Natl. Acad. Sci. USA 93:5824-5829), where site-specific mutation of the gene for the large subunit of ADPGPPase, using a transposable element Ds system, led to an insertion mutation of ADPGPPase which had decreased sensitivity to the ADPGPPase inhibitor, phosphate, as well as increased seed weight.
As regards to the use of wild species of Lycopersicon for the modification of carbohydrate metabolism in tomatoes, as described in U.S. patent application Ser. No. 08/530,216, although the fructose to glucose ratio in L. hirsutum is high, the actual amount of fructose and glucose is very low. Recombination of the genetic trait of fructose to glucose ratio, together with the trait of high glucose and fructose levels from L. esculentum yielded the unobvious and desirable trait of high levels of hexose, together with the high ratio of fructose to glucose. However, L. hirsutum fruit accumulate only low amounts of starch, as compared to the cultivated, L. esculentum (Miron and Schaffer, 1991, Plant Physiology 95:623-627). Similarly, other wild species of Lycopersicon also accumulate little starch (i.e., L chmielewskii, Yelle et at. 1988. Plant Physiology 87:737-740). Thus, the prior art has never expected or considered the use of wild tomatoes as a possible source of genetic variability for the increase in starch accumulation.
The present invention seeks to provide selection strategies for tomatoes with high starch content in the young fruit and subsequent high soluble solids in the mature fruit.
There is thus provided in accordance with a preferred embodiment of the present invention a method for controlling starch synthesis in tomatoes including providing a population of plants derived from interspecific crosses of Lycopersicon spp. with Lycopersicon esculenum genotypes, and selecting individuals of the population that each contain an allele of a gene that increases starch synthesis, the gene originating from the Lycopersicon spp.
In accordance with a preferred embodiment of the present invention the step of selecting includes selecting individuals that each contain the allele of the gene that encodes for an enzyme that catalyzes a metabolic step in starch synthesis.
Further in accordance with a preferred embodiment of the present invention the step of selecting includes selecting individuals that each contain the allele of the gene that encodes for a subunit of ADP-glucose pyrophosphorylase (ADPGPPase).
Still further in accordance with a preferred embodiment of the present invention the step of selecting includes selecting individuals that each contain the allele of the gene that encodes for a Lycopersicon hirsutum-derived subunit of ADPGPPase.
Additionally in accordance with a preferred embodiment of the present invention the step of selecting includes selecting by using a molecular marker for the gene.
In accordance with a preferred embodiment of the present invention the molecular marker is diagnostic for [includes step of selecting includes] a Lycopersicon hirsutum-derived large subunit (LS1) of ADPGPPase.
Further in accordance with a preferred embodiment of the present invention the step of selecting includes selecting by measuring activity of the enzyme in young fruit and selecting those young fruit with high activity of the enzyme.
Still further in accordance with a preferred embodiment of the present invention the step of selecting includes selecting by measuring ADPGPPase activity of the young fruit, and selecting those young fruit with high ADPGPPase activity.
In accordance with a preferred embodiment of the present invention the Lycopersicon spp. includes a Lycopersicon spp. of green-fruit Eriopersicon subgenus. Preferably the Lycopersicon spp. includes Lycopersicon hirsutum. 
There is also provided in accordance with a preferred embodiment of the present invention a method of producing genetically transformed plants which have elevated starch content, including the steps of inserting into the genome of a plant cell a recombinant double stranded DNA molecule including a selected promoter, a structural DNA sequence that causes the production of an RNA sequence which encodes the above described ADPGPPase LS1 protein, obtaining transformed plant cells, and regenerating from the transformed plant cells genetically transformed plants with elevated starch content.
In accordance with a preferred embodiment of the present invention the plant cell is selected from the group consisting of a tomato cell, a potato cell, a cell from a solanaceous plant, a legume cell, and a grain crop cell.
Further in accordance with a preferred embodiment of the present invention the promoter is selected from the group consisting of an immature fruit promoter, a tuber promoter, and a seed promoter.
Still further in accordance with a preferred embodiment of the present invention the step of regenerating includes regenerating genetically transformed plants with elevated starch content in an immature fruit
In accordance with a preferred embodiment of the present invention the step of regenerating includes regenerating genetically transformed plants with elevated starch content in a tuber.
Further in accordance with a preferred embodiment of the present invention the step of regenerating includes regenerating genetically transformed plants with elevated starch content in a seed.
Still further in accordance with a preferred embodiment of the present invention the methods of the present invention also include the step of propagating the individuals of the population or the genetically transformed plants. The propagating may be by vegetative propagation or by seed, for example.
There are also provided in accordance with a preferred embodiment of the present invention a plant produced according to any of the methods of the present invention, a fruit produced by such a plant, and a seed which when grown yields such a plant.